Spotlight Review

Leukemia (2011) 25, 193–200; doi:10.1038/leu.2010.197; published online 16 September 2010

Corrected online: 12 November 2010

There is a Erratum (1 February 2011) associated with this article.

Do we have to kill the last CML cell?

D M Ross1,2, T P Hughes1 and J V Melo1

  1. 1Department of Haematology, SA Pathology Centre for Cancer Biology, University of Adelaide, Adelaide, Australia
  2. 2Department of Haematology and Genetic Pathology, School of Medicine, Flinders University, Adelaide, Australia

Correspondence: Professor JV Melo, Department of Haematology, SA Pathology Centre for Cancer Biology, Frome Road, Adelaide, SA 5000, Australia. E-mail: junia.melo@health.sa.gov.au

Received 4 December 2009; Revised 27 July 2010; Accepted 5 August 2010; Corrected online 12 November 2010; Published online 16 September 2010.

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Abstract

Previous experience in the treatment of chronic myeloid leukaemia (CML) has shown that the achievement of clinical, morphological and cytogenetic remission does not indicate eradication of the disease. A complete molecular response (CMR; no detectable BCR–ABL mRNA) represents a deeper level of response, but even CMR is not a guarantee of elimination of the leukaemia, because the significance of CMR is determined by the detection limit of the assay that is used. Two studies of imatinib cessation in CMR are underway, cumulatively involving over 100 patients. The current estimated rate of stable CMR after stopping imatinib is approximately 40%, but the duration of follow-up is relatively short. The factors that determine relapse risk are yet to be identified. The intrinsic capacity of any residual leukaemic cells to proliferate following the withdrawal of treatment may be important, but there may also be a role for immunological suppression of the leukaemic clone. No currently available test can formally prove that the leukaemic clone is eradicated. Here we discuss the sensitive measurement of minimal residual disease, and speculate on the biology of BCR–ABL-positive cells that may persist after effective therapy of CML.

This article has been corrected since Advance Online Publication and an erratum is also printed in this issue

Keywords:

chronic myeloid leukaemia; minimal residual disease; PCR; BCR–ABL; imatinib mesylate

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Introduction

As the sensitivity of cytogenetic and PCR-based methods for the monitoring of chronic myeloid leukaemia (CML) has improved over time, it has become evident that the achievement of clinical, morphological and cytogenetic remission does not indicate eradication of the disease. For months or years after achieving a complete cytogenetic response (absence of Philadelphia (Ph)-positive cells in at least 20 bone marrow metaphase cells) the majority of CML patients have measurable disease by real-time reverse transcriptase quantitative PCR (RQ-PCR), and would relapse if imatinib treatment were withdrawn.1, 2 However, in the majority of patients who respond to imatinib there is a progressive decline in BCR–ABL mRNA over time, so that after several years of treatment an increasing number of chronic-phase CML patients will achieve a complete molecular response (CMR), with no BCR–ABL mRNA detectable by RQ-PCR. Whether the CML clone has been eradicated in any of these patients is a question of increasing clinical and scientific importance. The complete elimination of CML would require CMR, but CMR is not a guarantee of eradication of the leukaemia, because the significance of CMR is determined by the detection limit of the assay that is used.

Leukaemic cells can be identified by the presence of the Ph chromosome on cytogenetic analysis and/or the BCR–ABL fusion gene as detected by PCR. This enables a quantitative assessment of the burden of leukaemia and its response to treatment. The primary method of quantification of minimal residual disease (MRD) is RQ-PCR. Results are expressed as a ratio of BCR–ABL to a control gene. The RQ-PCR method and the choice of control gene differ between laboratories, so that the median baseline BCR–ABL:control gene ratio is not the same in all laboratories, making it difficult to compare results between centres. In order to overcome this difficulty, an international collaborative effort has led to the development of an international scale for the reporting of BCR–ABL mRNA levels, in which each result is normalized using a laboratory-specific conversion factor.3, 4, 5 For instance, on the international scale, a 2-log reduction from the median pre-treatment BCR–ABL level (determined in a pool of patients) is 1%. On the assumption that almost 100% of the cells are leukaemic at diagnosis, a BCR–ABL level of 1% may be viewed as indicating that the proportion of residual leukaemic cells is approximately 1% of that present at diagnosis.

Both on imatinib and on interferon-α (IFN) treatment, the achievement of a deeper cytogenetic response is associated with improved progression-free survival.6, 7 Within 18 months a complete cytogenetic response is observed in around 80% of the patients treated up-front with imatinib, and yet, even in this low-risk group, it has been demonstrated that a further reduction of the level of residual disease to less than or equal to0.1% BCR–ABL, termed a major molecular response (MMR), is associated with an improvement in progression-free survival.8 A proposed definition of CMR requires undetectable BCR–ABL transcripts in an RQ-PCR assay with a calculated sensitivity of at least 1.5 log below the level of MMR, and confirmed on subsequent testing.9 In a subset of patients in the International Randomised Study of Interferon versus STI571 (IRIS) study,6 loss of MMR was observed in 0/18 patients in CMR versus 6/22 (27%) of those in MMR who still had detectable BCR–ABL,9 suggesting that CMR confers a more stable response. However, as the progression-free survival of patients in MMR is close to 100%,8 it may be impossible to demonstrate that patients in CMR have a survival advantage over patients in MMR with detectable BCR–ABL, even if CMR does confer a better prognosis. Most of the available evidence suggests that prognosis improves as the level of MRD falls. It has not been proven that CMR confers a better prognosis than MMR, but this makes sense from first principles.

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Measuring the level of MRD

In RQ-PCR, the target gene, BCR–ABL, is reverse transcribed in parallel with a control gene, such as BCR. The number of copies of the control gene that can be detected in an individual sample will vary according to the amount of sample, the sample quality, and the efficiency of the reverse transcription reaction and the real-time PCR. The estimated limit of detection of RQ-PCR varies between laboratories, but is generally in the range of 10−4–10−5. Some laboratories perform nested PCR if BCR–ABL is not detectable in RQ-PCR, and have found that negative nested PCR represents a more stringent definition of CMR.10 Yet, other laboratories (including our own11) have found no improvement in the limit of detection of RQ-PCR using the conventional nested PCR for amplification of BCR–ABL complementary DNA.12 Such methodological differences highlight the need for a standardized definition of CMR for use in clinical trials.

Regardless of the PCR methodology in use, a major determinant of the sensitivity of the assay is the amount of patient material that is sampled.13, 14 This can be illustrated by considering the level of residual disease in complete cytogenetic response. If the Ph chromosome is not detected in 20 metaphase cells, one might assume that the level of MRD is <5%. Allowing for sampling error, which can be modelled using Poisson statistics, there is a 95% probability that the true level of MRD is <14%. The lower limit of detection can be improved simply by increasing the number of cells examined.

Virtually all patients with chronic-phase CML express one or both of the common e13a2 and e14a2 BCR–ABL mRNA transcripts. In any type of reverse transcriptase PCR this results in a risk of cross-contamination between patient samples when batches of samples containing variable levels of BCR–ABL are processed together. If stringent controls are in place to exclude mRNA cross-contamination, it is possible for a highly sensitive nested PCR method to achieve a sensitivity well below that of conventional RQ-PCR,15 but such methods are not practical for routine clinical use. Furthermore, the finding of occasional BCR–ABL mRNA transcripts in the blood of normal individuals15, 16 poses a limit to the specificity of highly sensitive PCR methods for the monitoring of MRD based on mRNA. This has led us and others to investigate the use of patient-specific BCR–ABL genomic DNA breakpoints as an investigational test to improve the lower limit of detection of MRD.17, 18, 19, 20 In order to perform patient-specific DNA PCR in CML, it is necessary to identify the BCR–ABL fusion region and to design primers for each patient to ensure specificity for the unique fusion sequence (Figure 1). The use of a patient-specific DNA sequence as the marker of the leukaemic clone is well established in acute lymphoblastic leukaemia, in which the target sequence is either the immunoglobulin gene or the T cell receptor gene rearrangement.21

Figure 1.
Figure 1 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Patient-specific DNA PCR for genomic BCR–ABL. A method for the detection of the BCR–ABL fusion sequence is shown. In most cases the BCR and ABL1 breakpoints are intronic and the region containing the fusion sequence is spliced out of the mRNA. Quantification of the patient-specific BCR–ABL DNA sequence is based on real-time PCR using a nested primer and probe set.

Full figure and legend (254K)

Sensitive PCR methods, including optimized RQ-PCR, are capable of detecting a single copy of BCR–ABL, if there is a copy present in the sample.22 However, regardless of the method used to quantify the level of MRD, it is impossible formally to demonstrate complete eradication of the CML clone, because it is not possible to test every single haematopoietic cell in the body. Serial MRD measurements during imatinib treatment may be used to extrapolate an approximate level of MRD after BCR–ABL is no longer detectable, but the accuracy of kinetic modelling is dependent on assumptions made about the rate of depletion of leukaemic cells over time. Ultimately, if the level of BCR–ABL is below the limit of detection, the only way to determine whether there is persistent residual disease is to withdraw therapy and monitor closely for evidence of molecular relapse. If molecular relapse occurs after the withdrawal of therapy, it can be concluded that MRD was present. On the other hand, if CMR is maintained in the long term, both leukaemia eradication and continuing suppression of residual leukaemia are plausible explanations.

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Kinetics of response to imatinib treatment

A plot of BCR–ABL RQ-PCR data from imatinib-treated chronic-phase CML patients reveals a biphasic response.23 The first phase of the imatinib response has a steep downward gradient, reflecting rapid clearance of mature CML progeny, and is associated with the achievement of haematological and cytogenetic response. The second phase has a shallow gradient and is thought to reflect gradual depletion of the CML granulocyte-macrophage precursor (GMP) pool.24 The latter phenomenon may be prognostically important, because it is in the GMP fraction of CML cells that transformation to myeloid blast crisis is thought to occur.25

The response to imatinib varies considerably between chronic-phase CML patients, despite the apparent homogeneity of the disease. Patients with a low-risk Sokal score at diagnosis, for instance, are more likely to achieve an MMR.8, 26, 27 Around 40% of the patients achieve an MMR within the first year8 and only 5% of patients achieve a CMR within the first 2 years,9 even though the rate of CMR reaches 40–50% after 5 years of imatinib treatment. CML progenitors are relatively resistant to imatinib,28, 29 and their slow depletion during imatinib treatment may reflect the induction of apoptosis in a manner that is dependent on the cell cycle. Beyond the second year of treatment the reduction in BCR–ABL mRNA is <0.5 log per annum in the majority of patients.9, 24 On the assumption that patients enter CMR with an MRD burden of the order of 106 cells, one could extrapolate that few patients would achieve eradication of the leukaemic clone, until after at least 10 years in CMR on continuing imatinib treatment.

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Eradication of CML stem cells

Extensive studies on CML stem cells in vitro have shown multiple pathways of resistance to tyrosine kinase inhibitors.28 These include decreased intracellular uptake and retention of cytotoxic drugs and tyrosine kinase inhibitors,28 and resistance to apoptosis.30, 31 Decreased expression of human leukocyte antigen co-stimulatory molecules and targets of adaptive immunity (such as myeloid granule proteins) might also protect stem cells from immune surveillance.32, 33 Many characteristics are shared by leukaemic and normal stem cells, making it difficult to develop a stem cell targeted therapy that is effective against CML and relatively non-toxic. Treatment with imatinib and other ABL kinase inhibitors in vitro results in an increase in quiescent CML stem cells that retain proliferative capacity when treatment is withdrawn.34, 35 Various factors that may contribute to the persistence of leukaemia after imatinib treatment are summarized in Figure 2. These data would lead us to predict that imatinib treatment must be continued indefinitely.

Figure 2.
Figure 2 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Some factors that might contribute to the persistence of MRD in CML. A wide range of factors intrinsic to the leukaemic clone or intrinsic to the patient (constitutional factors) may influence the response to therapy and the ultimate persistence or eradication of the leukaemic clone. These factors are summarized in this schematic diagram. Pharmacokinetics: increased bioavailability of imatinib or decreased clearance may enhance systemic drug exposure and kinase inhibition. Imatinib absorption in the gut is influenced by ATP-binding cassette sub-family B member 1 (ABCB1) and ATP-binding cassette sub-family G member 2 (ABCB2) and hepatic clearance is mediated by cytochrome P450 enzymes.80, 81 Cellular influx/efflux: the intracellular concentration of imatinib is dependent on the balance between the influx and efflux of the drug. Imatinib influx is organic cation transporter-1 dependent and organic cation transporter-1 activity varies between patients.82 Imatinib may be a substrate of the efflux proteins, ABCB1 and ABCB2.83, 84 Immunity: CML cells may evade immune recognition by downregulation of HLA molecules or leukaemia-associated antigens, or by downregulation of T cell co-stimulatory signals. CTLs and NK cells may be cytotoxic against CML cells.85 Autocrine/paracrine growth factors: CML cells produce cytokines that have anti-apoptotic effects.30, 31 Stromal homing: CML cells have abnormal interactions with tissue stroma, and stromal interactions may protect leukaemic cells from apoptosis.86, 87 BCR–ABL expression: high expression of BCR–ABL may be observed in advanced-phase CML88 and in CML stem cells,28 and may be associated with resistance to imatinib treatment.

Full figure and legend (68K)

In contrast, two studies of kinetic modelling found that CML might be eradicated by prolonged imatinib treatment. In one model, stem cells were depleted on the basis that susceptibility to apoptosis in response to imatinib is restored as quiescent stem cells enter the cell cycle.24 Long-term stem cells enter the cell cycle infrequently, and were therefore depleted very gradually in the model. A second study reached the same conclusion, but for a different reason.36 The authors incorporated in their model the stochastic process of stem cell exhaustion. Put simply, each time a precursor cell divides it gives rise to two daughter cells and each of these daughter cells will be committed either to differentiation or to self-renewal. The probability of each of these ‘choices’ is dependent upon the position of the cell in the haematopoietic hierarchy; more mature precursor cells are more likely to commit to differentiation. Although, on average, a sufficient number of stem cells will commit to self-renewal, in the case of a single leukaemic stem cell it is possible that both progeny should commit to differentiation and the leukaemic stem cell would then be extinct. Based on assumptions about stem cell biology the authors constructed a mathematical model of CML that incorporated a stochastic process of stem cell renewal versus differentiation. In most iterations of the model the CML stem cell pool was already exhausted before the ‘patient’ commenced treatment and the proportion of ‘patients’ in whom the CML stem cell pool became extinct rose progressively during imatinib treatment. As with any disease model, the validity of these predictions is dependent upon the accuracy of the assumptions from known disease biology. Considering this caveat, we have at least two potential explanations for why a treatment that is apparently cytostatic to stem cells could still have curative potential in CML.

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Transplantation

Allogeneic stem cell transplantation remains the only therapy for CML that has been shown to achieve a durable CMR, with long-term follow-up in excess of a decade, described by Goldman as an ‘operational cure’.37 Myeloablative conditioning for transplantation may result in a profound reduction in leukaemic burden, but it appears that this alone is not sufficient to achieve long-term control of the leukaemia. A graft-versus-leukaemia immunological effect contributes to the long-term success of the treatment, as evidenced by the higher relapse risk in syngeneic transplants than in allografts, and by the efficacy of response to donor lymphocyte infusion.

Even in allograft recipients it is uncertain whether MRD has been eradicated. Very late relapses may occur even after decades38 and low levels of BCR–ABL mRNA may be detected intermittently without overt relapse.39, 40 In order to determine whether rare BCR–ABL transcripts truly reflect persistence of the original CML clone, we performed patient-specific BCR–ABL DNA PCR in patients in long-term remission post-allograft. These results showed that in most cases occasional detectable BCR–ABL transcripts were not associated with detectable BCR–ABL DNA, indicating that these low-level positive RQ-PCR results are not reliable indicators of residual disease. Although the majority of allograft patients in long-term remission had no detectable BCR–ABL DNA, it remains possible that MRD persists below the limit of detection.

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Interferon treatment

The mechanism of action of IFN in CML is unclear, but may involve antiproliferative effects,41 altered stem cell self-renewal,42 pro-apoptotic effects43 and augmentation of naturally occurring immunological responses against leukaemia-associated antigens.44, 45, 46 Complete cytogenetic responses occur in up to 20–30% of chronic-phase patients treated with IFN (±cytarabine) and such patients have a significant improvement in progression-free survival.47 However, only a small minority of IFN-treated patients achieve a stable CMR.

Cessation of IFN treatment in the minority of patients who achieve a deep molecular response may be associated with stable MMR for a period in excess of 9 years off treatment.48, 49 Other IFN-treated patients have been reported in patients who remained in stable complete cytogenetic response after cessation of IFN.50, 51 It seems likely that this stable level of MRD after cessation of IFN treatment may reflect ongoing immunological suppression of CML. IFN increases the expression of known leukaemia-associated antigens and is associated with the development of T cell responses against such antigens.44, 45

IFN may induce proliferation of CML stem cells,42 thus contributing to stem cell exhaustion and providing a rationale for combination therapy with tyrosine kinase inhibitors, which are otherwise ineffective against quiescent CML stem cells. Extending this hypothesis, Burchert et al.52 used combination therapy with IFN and imatinib and then stopped imatinib, using IFN as maintenance treatment. Cytotoxic T lymphocytes (CTLs) reactive against a leukaemia-associated antigen were observed more frequently after imatinib was stopped, and there was a suggestion that failure to develop this response was associated with an increase in the level of MRD.

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Cessation of imatinib treatment

Mathematical modelling of relapsing CML using RQ-PCR data and model assumptions from stem cell biology predicts a rapid increase in BCR–ABL when treatment is withdrawn.23, 24 In this model such increase is due to the unchecked proliferation of progeny derived from a pool of CML GMP precursors. The time taken for MRD to rise to a level that results in molecular relapse will depend on the proliferative capacity of the GMPs and on the size of the GMP pool (as well as on the lower detection limit of the assay). Given the high proliferative capacity of CML cells, it might be predicted that the size of the GMP pool at the time of imatinib cessation would be the primary determinant of the time to relapse. With an average relapse gradient of 0.02 log per day23 the predicted time for unchecked proliferation of a single leukaemic cell to result in loss of CMR would be 10–12 months.

There is currently limited information on the outcome of CML patients after the cessation of imatinib treatment in CMR. A cohort of 12 patients was reported by the French CML Intergroup: those patients had a minimum duration of CMR of 2 years, and were monitored monthly with RQ-PCR after imatinib cessation. A total of six patients relapsed within the first 6 months, while the remaining 50% of patients were in stable CMR.53 Only two patients in this pilot study had received imatinib treatment de novo and both of these patients relapsed. The remaining 10 patients had all received IFN therapy previously. A case report from the MD Anderson Cancer Center detailed for the first time stable CMR for more than 6 months after imatinib cessation in a single patient treated with imatinib de novo.54

There are now at least two studies underway examining the outcome of chronic-phase CML patients following imatinib cessation in stable CMR. Interim results of the follow-up study from the French CML intergroup were reported in 2009:55 with 69 patients enrolled and a median follow-up of 17 months, the relapse rate was 55%. Importantly, the relapse rate was similar in those patients who received imatinib de novo (54%) and those treated with imatinib after the previous IFN therapy (56%). A similar study, currently having enrolled 35 patients, is being conducted by the Australasian Leukaemia & Lymphoma Group.19 At the most recent interim analysis (May 2010), the estimated molecular relapse rate was 60% with a median follow-up of 21 months. A total of 12 patients were in continuous CMR without any therapy for 12 months or more. Across both studies, only occasional relapses have occurred more than 6 months after stopping imatinib. The latest relapse to date was 24 months after imatinib cessation (in the Australasian study). It should be emphasized that in both of these studies relapse was defined as detectable BCR–ABL mRNA and, to the best of our knowledge, none of these patients has experienced a cytogenetic or haematological relapse of CML. Resumption of imatinib treatment after confirmation of molecular relapse typically restores CMR within 6 months.

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Persistent MRD might not always result in relapse

There is an apparent dichotomy in imatinib-treated patients between early relapse and sustained remission, and we hypothesized that this could reflect differing levels of MRD, with those patients in remission having a lower level of MRD, or even having eradicated the CML clone. In a subset of 18 patients in the Australasian imatinib cessation study we used a sensitive, patient-specific DNA PCR method to show that a measurable level of MRD was present in most patients in CMR (Figure 1).19 This intronic sequence can then be amplified using a forward primer in BCR and a reverse primer in ABL1. The measured level of MRD in these patients was in the range of 0.5–1.5 log below the conventional RQ-PCR threshold for CMR. This is consistent with extrapolated RQ-PCR data, which would predict a level of MRD of around 1 log below the detection limit of RQ-PCR in most patients after 2 years in a stable CMR.

Several hypotheses might explain why a pool of viable leukaemic cells should not cause relapse. In transplantation models of leukaemia there may be a critical cell dose required to initiate leukaemia, but this probably reflects the rarity of true stem cells in a mixed population of leukaemic cells, rather than the need for a critical initiating mass of leukaemic progenitors. Second, the leukaemic cells that survive after imatinib treatment might be functionally defective or terminally differentiated (for example, memory B cells). An alternative explanation, for which there is currently the most evidence, is that MRD is suppressed below the limit of detection by an ongoing immunological response against the leukaemic clone.

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Immunological control of CML

The best evidence for immunological suppression of the leukaemic clone in CML comes from patients with residual disease or relapse after allogeneic stem cell transplantation, supported by data from IFN-treated patients, as discussed above. There is a link between clinical response to IFN therapy and the emergence of CTLs specific for myeloid-associated antigens.44 CML patients whose CD34+ leukaemic cells expressed higher levels of the leukaemia-associated antigen, proteinase 3, had more indolent disease,33 raising the possibility that a more immunogenic leukaemia is more effectively suppressed by the host immune system. There is conflicting evidence as to whether similar CTL responses also occur during imatinib treatment.45, 56 In fact, rare CTLs against CML-associated antigens can be found even in the blood of normal individuals.57 This is perhaps not surprising, given that many of the antigens associated with a CTL response in CML (and other myeloid malignancies) are widely expressed in normal cells.58 Leukaemia-specific autologous CTLs can be elicited by peptide vaccination using the amino-acid sequence of the BCR–ABL junction,57, 59, 60 and there is preliminary evidence that an immunological response to vaccination might result in a clinically significant reduction in the level of MRD.60, 61

Another interesting observation from allograft patients is the response to imatinib when used to treat relapse post-allograft: with or without donor lymphocyte infusion a stable CMR is typically achieved very quickly.62, 63 This suggests that, after allografting, the suppression of MRD by imatinib might help to restore effective immunological control. Several studies have shown that CTL responses against leukaemia-associated antigens emerged or were augmented in response to a reduction in disease burden.44, 56, 64 This finding has parallels in a mouse model of the immune response to chronic infection,65 from which the authors concluded that a reduction in the antigenic burden (by chemotherapy) might be necessary to prevent functional impairment of the host immune response against a chronic pathogen. The importance of functional impairment of the anti-leukaemic CTL response was further demonstrated in a mouse model of CML in which upregulation of the programmed cell death-1 receptor was associated with immune exhaustion and disease progression.66 Manipulation of the co-stimulatory molecules that contribute to immune exhaustion might in the future open up a novel class of therapeutics for CML.

Imatinib suppresses T cell function67 and could potentially impair immune surveillance against CML. The more potent ABL kinase inhibitors, nilotinib and dasatinib, are both available in many countries for patients who have failed imatinib treatment, and clinical trials of de novo treatment with these drugs have shown promising responses. In the future it is possible that the use of more potent inhibitors of BCR–ABL will increase the proportion of patients who achieve a stable CMR. However, if immunological surveillance is important in determining the stability of CMR in imatinib-treated CML patients, then the more potent T cell immunosuppressive effects of dasatinib68, 69, 70 and nilotinib71 might alter the risk of relapse when therapy is withdrawn. Intriguingly, despite such theoretical concerns, the emergence of increased numbers of large granular lymphocytes in the peripheral blood (CTL or natural killer cell type) has been observed during dasatinib treatment and appears to be associated with improved CML responses.72

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Disease resistance and progression

The primary aim of all treatments for chronic-phase CML is to prevent progression to accelerated phase or blast crisis. After transformation, the response to therapy is poor and blast crisis carries a life expectancy of less than 12 months.73, 74 The mechanisms of transformation are complex, but in most cases the acquisition of new genetic abnormalities is essential for the activation of pathways that confer resistance to treatment.75 DNA damage is a stochastic process, so a smaller number of ‘at-risk’ cells must translate into a lower probability that a resistant mutation will arise. This theoretical consideration is supported by the strong relationship between the level of MRD achieved during treatment and the subsequent risk of disease progression.6, 7, 8

BCR–ABL is itself mutagenic: the genome of a CML cell has an increased rate of mutation due to a combination of increased damage by reactive oxygen species76 and defective DNA repair.77, 78, 79 These abnormalities are at least partly dependent on BCR–ABL kinase activity, so the effective inhibition of BCR–ABL by imatinib would be expected to reduce the risk of acquiring a new mutation. Imatinib treatment of CML cells in vitro results in the accumulation of quiescent CML precursors that are resistant to imatinib.28, 29 These cells are not in active cell cycle, and it is unclear whether or not they are protected from DNA damage so long as they remain quiescent. BCR–ABL mRNA is expressed in CML stem cells34, 35 and BCR–ABL kinase activity (measured as protein substrate phosphorylation) may persist in CD34+ CD38− CML stem cells exposed to imatinib ex vivo.35 If mutagenesis is not suppressed in CML precursors that survive treatment, then a small risk of disease progression will persist as long as there are any viable CML cells.

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Do we have to kill the last CML cell?

At present, this must remain a rhetorical question, as we do not (and probably never will) have the ability to measure the ‘last CML cell’. This question can really be answered only by the careful observation of patients who stop therapy in CMR; however, if relapse can occur 20 years after an allograft, then how long must we wait for an answer?

Any persistence of BCR–ABL in viable leukaemic stem cells or early progenitors must result in a lasting risk of relapse and disease progression, and therefore complete eradication should be our goal. If the risk of disease progression is as small in imatinib-treated patients as it is in patients in long-term remission after allografting, then a pragmatic response may be that eradication is not necessary. If it is true that stem cell exhaustion is common in CML, as predicted by Lenaerts et al.,36 then perhaps we need only to suppress the disease for long enough and it will burn itself out.

Two ongoing studies of imatinib cessation suggest that around 40% of patients with a durable CMR on imatinib treatment may remain in CMR when treatment is withdrawn, and it is possible that CML has been eradicated in some of these patients. A longer follow-up of patients in stable CMR without imatinib treatment is needed to determine whether these patients can be considered to have been cured. At least it is clear that early relapse is not inevitable when imatinib treatment is withdrawn from carefully selected patients. Further progress on (1) the measurement of low levels of MRD; (2) better understanding of the pathways in leukaemic cells that determine response to therapy and persistence of MRD; and (3) definition of the role of immunological reactivity against CML is needed. These studies might enable us prospectively to identify imatinib-treated patients who have a low risk of relapse when treatment is stopped.

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Conflict of interest

The authors declare no conflict of interest.

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References

  1. Mauro MJ, Druker BJ, Maziarz RT. Divergent clinical outcome in two CML patients who discontinued imatinib therapy after achieving a molecular remission. Leuk Res 2004; 28 (Suppl 1): S71–S73. | Article | PubMed | ISI | ChemPort |
  2. Cortes J, O'Brien S, Kantarjian H. Discontinuation of imatinib therapy after achieving a molecular response. Blood 2004; 104: 2204–2205. | Article | PubMed | ISI | ChemPort |
  3. Branford S, Cross NC, Hochhaus A, Radich J, Saglio G, Kaeda J et al. Rationale for the recommendations for harmonizing current methodology for detecting BCR-ABL transcripts in patients with chronic myeloid leukaemia. Leukemia 2006; 20: 1925–1930. | Article | PubMed | ISI | ChemPort |
  4. Branford S, Fletcher L, Cross NC, Muller MC, Hochhaus A, Kim DW et al. Desirable performance characteristics for BCR-ABL measurement on an international reporting scale to allow consistent interpretation of individual patient response comparison of response rates between clinical trials. Blood 2008; 112: 3330–3338. | Article | PubMed | ISI | ChemPort |
  5. Hughes T, Deininger M, Hochhaus A, Branford S, Radich J, Kaeda J et al. Monitoring CML patients responding to treatment with tyrosine kinase inhibitors: review recommendations for harmonizing current methodology for detecting BCR-ABL transcripts kinase domain mutations for expressing results. Blood 2006; 108: 28–37. | Article | PubMed | ISI | ChemPort |
  6. O'Brien SG, Guilhot F, Larson RA, Gathmann I, Baccarani M, Cervantes F et al. Imatinib compared with interferon low-dose cytarabine for newly diagnosed chronic-phase chronic myeloid leukemia. N Engl J Med 2003; 348: 994–1004. | Article | PubMed | ISI | ChemPort |
  7. Guilhot F, Chastang C, Michallet M, Guerci A, Harousseau JL, Maloisel F et al. Interferon alfa-2b combined with cytarabine versus interferon alone in chronic myelogenous leukemia. French Chronic Myeloid Leukemia Study Group. N Engl J Med 1997; 337: 223–229. | Article | PubMed | ISI | ChemPort |
  8. Hughes TP, Kaeda J, Branford S, Rudzki Z, Hochhaus A, Hensley ML et al. Frequency of major molecular responses to imatinib or interferon alfa plus cytarabine in newly diagnosed chronic myeloid leukemia. N Engl J Med 2003; 349: 1423–1432. | Article | PubMed | ISI | ChemPort |
  9. Branford S, Seymour JF, Grigg A, Arthur C, Rudzki Z, Lynch K et al. BCR-ABL messenger RNA levels continue to decline in patients with chronic phase chronic myeloid leukemia treated with imatinib for more than 5 years approximately half of all first-line treated patients have stable undetectable BCR-ABL using strict sensitivity criteria. Clin Cancer Res 2007; 13: 7080–7085. | Article | PubMed | ISI | ChemPort |
  10. Muller MC, Gattermann N, Lahaye T, Deininger MW, Berndt A, Fruehauf S et al. Dynamics of BCR-ABL mRNA expression in first-line therapy of chronic myelogenous leukemia patients with imatinib or interferon alpha/ara-C. Leukemia 2003; 17: 2392–2400. | Article | PubMed | ISI | ChemPort |
  11. Ross DM, Watkins DB, Hughes TP, Branford S. Reverse transcription with random pentadecamer primers improves the detection limit of a quantitative PCR assay for BCR-ABL transcripts in chronic myeloid leukemia: implications for defining sensitivity in minimal residual disease. Clin Chem 2008; 54: 1568–1571. | Article | PubMed | ISI
  12. Cross NC, Hughes TP, Feng L, O'Shea P, Bungey J, Marks DI et al. Minimal residual disease after allogeneic bone marrow transplantation for chronic myeloid leukaemia in first chronic phase: correlations with acute graft-versus-host disease relapse. Br J Haematol 1993; 84: 67–74. | Article | PubMed | ISI | ChemPort |
  13. Melo JV, Yan XH, Diamond J, Lin F, Cross NC, Goldman JM. Reverse transcription/polymerase chain reaction (RT/PCR) amplification of very small numbers of transcripts: the risk in misinterpreting negative results. Leukemia 1996; 10: 1217–1221. | PubMed | ISI | ChemPort |
  14. Ross DM, Branford S, Melo JV, Hughes TP. Reply to ‘What do we mean by sensitivity when we talk about detecting minimal residual disease?’ by Steinbach and Debatin. Leukemia 2009; 23: 819–820;author reply 820. | Article | PubMed | ISI
  15. Bose S, Deininger M, Gora-Tybor J, Goldman JM, Melo JV. The presence of typical and atypical BCR-ABL fusion genes in leukocytes of normal individuals: biologic significance implications for the assessment of minimal residual disease. Blood 1998; 92: 3362–3367. | PubMed | ISI | ChemPort |
  16. Biernaux C, Loos M, Sels A, Huez G, Stryckmans P. Detection of major bcr-abl gene expression at a very low level in blood cells of some healthy individuals. Blood 1995; 86: 3118–3122. | PubMed | ISI | ChemPort |
  17. Mattarucchi E, Spinelli O, Rambaldi A, Pasquali F, Lo Curto F, Campiotti L et al. Molecular monitoring of residual disease in chronic myeloid leukemia by genomic DNA compared with conventional mRNA analysis. J Mol Diagn 2009; 11: 482–487. | PubMed | ISI |
  18. Sobrinho-Simoes M, Wilczek V, Score J, Cross NC, Apperley JF, Melo JV. In search of the original leukemic clone in chronic myeloid leukemia patients in complete molecular remission after stem cell transplantation or imatinib. Blood 2010; 116: 1329–1335. | Article | PubMed | ISI
  19. Ross DM, Branford S, Seymour JF, Schwarer AP, Arthur C, Bartley PA et al. Patients with chronic myeloid leukaemia who maintain a complete molecular response after stopping imatinib treatment have evidence of persistent leukaemia by DNA PCR. Leukemia 2010; in press.
  20. Bartley PA, Ross DM, Latham S, Martin-Harris MH, Budgen B, Wilczek V et al. Sensitive detection quantification of minimal residual disease in chronic myeloid leukaemia using nested quantitative PCR for BCR-ABL DNA. Int J Lab Hematol 2010; e-pub ahead of print 10 May 2010.
  21. van der Velden VH, Panzer-Grumayer ER, Cazzaniga G, Flohr T, Sutton R, Schrauder A et al. Optimization of PCR-based minimal residual disease diagnostics for childhood acute lymphoblastic leukemia in a multi-center setting. Leukemia 2007; 21: 706–713. | Article | PubMed | ISI | ChemPort |
  22. Rawer D, Borkhardt A, Wilda M, Kropf S, Kreuder J. Influence of stochastics on quantitative PCR in the detection of minimal residual disease. Leukemia 2003; 17: 2527–2528; author reply 2528–2531. | Article | PubMed | ISI | ChemPort |
  23. Michor F, Hughes TP, Iwasa Y, Branford S, Shah NP, Sawyers CL et al. Dynamics of chronic myeloid leukaemia. Nature 2005; 435: 1267–1270. | Article | PubMed | ISI | ChemPort |
  24. Roeder I, Horn M, Glauche I, Hochhaus A, Mueller MC, Loeffler M. Dynamic modeling of imatinib-treated chronic myeloid leukemia: functional insights clinical implications. Nat Med 2006; 12: 1181–1184. | Article | PubMed | ISI | ChemPort |
  25. Jamieson CH, Ailles LE, Dylla SJ, Muijtjens M, Jones C, Zehnder JL et al. Granulocyte-macrophage progenitors as candidate leukemic stem cells in blast-crisis CML. N Engl J Med 2004; 351: 657–667. | Article | PubMed | ISI | ChemPort |
  26. Forrest DL, Trainor S, Brinkman RR, Barnett MJ, Hogge DE, Nevill TJ et al. Cytogenetic molecular responses to standard-dose imatinib in chronic myeloid leukemia are correlated with Sokal risk scores duration of therapy but not trough imatinib plasma levels. Leuk Res 2009; 33: 271–275. | Article | PubMed | ISI | ChemPort |
  27. Larson RA, Druker BJ, Guilhot F, O'Brien SG, Riviere GJ, Krahnke T et al. Imatinib pharmacokinetics and its correlation with response safety in chronic-phase chronic myeloid leukemia: a subanalysis of the IRIS study. Blood 2008; 111: 4022–4028. | Article | PubMed | ISI | ChemPort |
  28. Jiang X, Zhao Y, Smith C, Gasparetto M, Turhan A, Eaves A et al. Chronic myeloid leukemia stem cells possess multiple unique features of resistance to BCR-ABL targeted therapies. Leukemia 2007; 21: 926–935. | Article | PubMed | ISI | ChemPort |
  29. Graham SM, Jorgensen HG, Allan E, Pearson C, Alcorn MJ, Richmond L et al. Primitive quiescent Philadelphia-positive stem cells from patients with chronic myeloid leukemia are insensitive to STI571 in vitro. Blood 2002; 99: 319–325. | Article | PubMed | ISI | ChemPort |
  30. Dorsey JF, Cunnick JM, Lanehart R, Huang M, Kraker AJ, Bhalla KN et al. Interleukin-3 protects Bcr-Abl-transformed hematopoietic progenitor cells from apoptosis induced by Bcr-Abl tyrosine kinase inhibitors. Leukemia 2002; 16: 1589–1595. | Article | PubMed | ISI | ChemPort |
  31. Holyoake TL, Jiang X, Jorgensen HG, Graham S, Alcorn MJ, Laird C et al. Primitive quiescent leukemic cells from patients with chronic myeloid leukemia spontaneously initiate factor-independent growth in vitro in association with up-regulation of expression of interleukin-3. Blood 2001; 97: 720–728. | Article | PubMed | ISI | ChemPort |
  32. Hirano N, Takahashi T, Ohtake S, Hirashima K, Emi N, Saito K et al. Expression of costimulatory molecules in human leukemias. Leukemia 1996; 10: 1168–1176. | PubMed | ISI | ChemPort |
  33. Yong AS, Szydlo RM, Goldman JM, Apperley JF, Melo JV. Molecular profiling of CD34+ cells identifies low expression of CD7 along with high expression of proteinase 3 or elastase as predictors of longer survival in patients with CML. Blood 2006; 107: 205–212. | Article | PubMed | ISI | ChemPort |
  34. Holyoake T, Jiang X, Eaves C, Eaves A. Isolation of a highly quiescent subpopulation of primitive leukemic cells in chronic myeloid leukemia. Blood 1999; 94: 2056–2064. | PubMed | ISI | ChemPort |
  35. Copland M, Hamilton A, Elrick LJ, Baird JW, Allan EK, Jordanides N et al. Dasatinib (BMS-354825) targets an earlier progenitor population than imatinib in primary CML but does not eliminate the quiescent fraction. Blood 2006; 107: 4532–4539. | Article | PubMed | ISI | ChemPort |
  36. Lenaerts T, Pacheco JM, Traulsen A, Dingli D. Tyrosine kinase inhibitor therapy can cure chronic myeloid leukemia without hitting leukemic stem cells. Haematologica 2010; 95: 900–907. | Article | PubMed | ISI
  37. Goldman J, Gordon M. Why do chronic myelogenous leukemia stem cells survive allogeneic stem cell transplantation or imatinib: does it really matter? Leuk Lymphoma 2006; 47: 1–7. | Article | PubMed | ISI | ChemPort |
  38. Clift RA, Appelbaum FR, Thomas ED. Treatment of chronic myeloid leukemia by marrow transplantation. Blood 1993; 82: 1954–1956. | PubMed | ISI | ChemPort |
  39. Kaeda J, O'Shea D, Szydlo RM, Olavarria E, Dazzi F, Marin D et al. Serial measurement of BCR-ABL transcripts in the peripheral blood after allogeneic stem cell transplantation for chronic myeloid leukemia: an attempt to define patients who may not require further therapy. Blood 2006; 107: 4171–4176. | Article | PubMed | ISI | ChemPort |
  40. Radich JP, Gehly G, Gooley T, Bryant E, Clift RA, Collins S et al. Polymerase chain reaction detection of the BCR-ABL fusion transcript after allogeneic marrow transplantation for chronic myeloid leukemia: results and implications in 346 patients. Blood 1995; 85: 2632–2638. | PubMed | ISI | ChemPort |
  41. Talpaz M, McCredie KB, Mavligit GM, Gutterman JU. Leukocyte interferon-induced myeloid cytoreduction in chronic myelogenous leukemia. Blood 1983; 62: 689–692. | PubMed | ISI | ChemPort |
  42. Essers MA, Offner S, Blanco-Bose WE, Waibler Z, Kalinke U, Duchosal MA et al. IFNalpha activates dormant haematopoietic stem cells in vivo. Nature 2009; 458: 904–908. | Article | PubMed | ISI | ChemPort |
  43. Selleri C, Sato T, Del Vecchio L, Luciano L, Barrett AJ, Rotoli B et al. Involvement of Fas-mediated apoptosis in the inhibitory effects of interferon-alpha in chronic myelogenous leukemia. Blood 1997; 89: 957–964. | PubMed | ISI | ChemPort |
  44. Molldrem JJ, Lee PP, Wang C, Felio K, Kantarjian HM, Champlin RE et al. Evidence that specific T lymphocytes may participate in the elimination of chronic myelogenous leukemia. Nat Med 2000; 6: 1018–1023. | Article | PubMed | ISI | ChemPort |
  45. Burchert A, Wolfl S, Schmidt M, Brendel C, Denecke B, Cai D et al. Interferon-alpha, but not the ABL-kinase inhibitor imatinib (STI571), induces expression of myeloblastin and a specific T-cell response in chronic myeloid leukemia. Blood 2003; 101: 259–264. | Article | PubMed | ISI | ChemPort |
  46. Molldrem JJ, Lee PP, Kant S, Wieder E, Jiang W, Lu S et al. Chronic myelogenous leukemia shapes host immunity by selective deletion of high-avidity leukemia-specific T cells. J Clin Invest 2003; 111: 639–647. | Article | PubMed | ISI | ChemPort |
  47. Guilhot F, Roy L, Guilhot J, Millot F. Interferon therapy in chronic myelogenous leukemia. Hematol Oncol Clin North Am 2004; 18: 585–603, viii. | Article | PubMed | ISI
  48. Mahon FX, Delbrel X, Cony-Makhoul P, Faberes C, Boiron JM, Barthe C et al. Follow-up of complete cytogenetic remission in patients with chronic myeloid leukemia after cessation of interferon alfa. J Clin Oncol 2002; 20: 214–220. | Article | PubMed | ISI
  49. Verbeek W, Konig H, Boehm J, Kohl D, Lange C, Heuer T et al. Continuous complete hematological and cytogenetic remission with molecular minimal residual disease 9 years after discontinuation of interferon-alpha in a patient with Philadelphia chromosome-positive chronic myeloid leukemia. Acta Haematol 2006; 115: 109–112. | Article | PubMed | ISI
  50. Bonifazi F, de Vivo A, Rosti G, Guilhot F, Guilhot J, Trabacchi E et al. Chronic myeloid leukemia and interferon-alpha: a study of complete cytogenetic responders. Blood 2001; 98: 3074–3081. | Article | PubMed | ISI | ChemPort |
  51. Kantarjian HM, O'Brien S, Cortes JE, Shan J, Giles FJ, Rios MB et al. Complete cytogenetic and molecular responses to interferon-alpha-based therapy for chronic myelogenous leukemia are associated with excellent long-term prognosis. Cancer 2003; 97: 1033–1041. | Article | PubMed | ISI | ChemPort |
  52. Burchert A, Muller MC, Kostrewa P, Erben P, Bostel T, Liebler S et al. Sustained molecular response with interferon alfa maintenance after induction therapy with imatinib plus interferon alfa in patients with chronic myeloid leukemia. J Clin Oncol 2010; 28: 1429–1435. | Article | PubMed | ISI | ChemPort |
  53. Rousselot P, Huguet F, Rea D, Legros L, Cayuela JM, Maarek O et al. Imatinib mesylate discontinuation in patients with chronic myelogenous leukemia in complete molecular remission for more than 2 years. Blood 2007; 109: 58–60. | Article | PubMed | ISI | ChemPort |
  54. Verma D, Kantarjian H, Jain N, Cortes J. Sustained complete molecular response after imatinib discontinuation in a patient with chronic myeloid leukemia not previously exposed to interferon alpha. Leuk Lymphoma 2008; 49: 353–1402. | Article | PubMed
  55. Mahon FX, Rea D, Guilhot F, Huguet F, Nicolini FE, Legros L et al. Discontinuation of imatinib therapy after achieving a complete molecular response in chronic myeloid leukemia patients. Blood 2009; 114: 353 (abstract no. 859). | ISI |
  56. Chen CI, Maecker HT, Lee PP. Development and dynamics of robust T-cell responses to CML under imatinib treatment. Blood 2008; 111: 5342–5349. | Article | PubMed | ISI
  57. Rezvani K, Grube M, Brenchley JM, Sconocchia G, Fujiwara H, Price DA et al. Functional leukemia-associated antigen-specific memory CD8+ T cells exist in healthy individuals and in patients with chronic myelogenous leukemia before and after stem cell transplantation. Blood 2003; 102: 2892–2900. | Article | PubMed | ISI | ChemPort |
  58. Barrett AJ, Rezvani K. Translational mini-review series on vaccines: Peptide vaccines for myeloid leukaemias. Clin Exp Immunol 2007; 148: 189–198. | Article | PubMed | ISI | ChemPort |
  59. Pinilla-Ibarz J, Cathcart K, Korontsvit T, Soignet S, Bocchia M, Caggiano J et al. Vaccination of patients with chronic myelogenous leukemia with bcr-abl oncogene breakpoint fusion peptides generates specific immune responses. Blood 2000; 95: 1781–1787. | PubMed | ISI | ChemPort |
  60. Rojas JM, Knight K, Wang L, Clark RE. Clinical evaluation of BCR-ABL peptide immunisation in chronic myeloid leukaemia: results of the EPIC study. Leukemia 2007; 21: 2287–2295. | Article | PubMed | ISI | ChemPort |
  61. Bocchia M, Gentili S, Abruzzese E, Fanelli A, Iuliano F, Tabilio A et al. Effect of a p210 multipeptide vaccine associated with imatinib or interferon in patients with chronic myeloid leukaemia and persistent residual disease: a multicentre observational trial. Lancet 2005; 365: 657–662. | PubMed | ISI | ChemPort |
  62. DeAngelo DJ, Hochberg EP, Alyea EP, Longtine J, Lee S, Galinsky I et al. Extended follow-up of patients treated with imatinib mesylate (gleevec) for chronic myelogenous leukemia relapse after allogeneic transplantation: durable cytogenetic remission and conversion to complete donor chimerism without graft-versus-host disease. Clin Cancer Res 2004; 10: 5065–5071. | Article | PubMed | ISI | ChemPort |
  63. Savani BN, Montero A, Kurlander R, Childs R, Hensel N, Barrett AJ. Imatinib synergizes with donor lymphocyte infusions to achieve rapid molecular remission of CML relapsing after allogeneic stem cell transplantation. Bone Marrow Transplant 2005; 36: 1009–1015. | Article | PubMed | ISI | ChemPort |
  64. Butt NM, Rojas JM, Wang L, Christmas SE, Abu-Eisha HM, Clark RE. Circulating bcr-abl-specific CD8+ T cells in chronic myeloid leukemia patients and healthy subjects. Haematologica 2005; 90: 1315–1323. | PubMed | ISI | ChemPort |
  65. Bustamante JM, Bixby LM, Tarleton RL. Drug-induced cure drives conversion to a stable and protective CD8+ T central memory response in chronic Chagas disease. Nat Med 2008; 14: 542–550. | Article | PubMed | ISI
  66. Mumprecht S, Schurch C, Schwaller J, Solenthaler M, Ochsenbein AF. Programmed death 1 signaling on chronic myeloid leukemia-specific T cells results in T-cell exhaustion and disease progression. Blood 2009; 114: 1528–1536. | Article | PubMed | ISI | ChemPort |
  67. Dietz AB, Souan L, Knutson GJ, Bulur PA, Litzow MR, Vuk-Pavlovic S. Imatinib mesylate inhibits T-cell proliferation in vitro and delayed-type hypersensitivity in vivo. Blood 2004; 104: 1094–1099. | Article | PubMed | ISI | ChemPort |
  68. Schade AE, Schieven GL, Townsend R, Jankowska AM, Susulic V, Zhang R et al. Dasatinib, a small-molecule protein tyrosine kinase inhibitor, inhibits T-cell activation and proliferation. Blood 2008; 111: 1366–1377. | Article | PubMed | ISI | ChemPort |
  69. Blake S, Hughes TP, Mayrhofer G, Lyons AB. The Src/ABL kinase inhibitor dasatinib (BMS-354825) inhibits function of normal human T-lymphocytes in vitro. Clin Immunol 2008; 127: 330–339. | Article | PubMed | ISI | ChemPort |
  70. Blake SJ, Lyons AB, Fraser CK, Hayball JD, Hughes TP. Dasatinib suppresses in vitro natural killer cell cytotoxicity. Blood 2008; 111: 4415–4416. | Article | PubMed | ISI | ChemPort |
  71. Blake SJ, Lyons AB, Hughes TP. Nilotinib inhibits the Src-family kinase LCK and T-cell function in vitro. J Cell Mol Med 2009; 13: 599–601. | Article | PubMed
  72. Mustjoki S, Ekblom M, Arstila TP, Dybedal I, Epling-Burnette PK, Guilhot F et al. Clonal expansion of T/NK-cells during tyrosine kinase inhibitor dasatinib therapy. Leukemia 2009; 23: 1398–1405. | Article | PubMed | ISI
  73. Sawyers CL, Hochhaus A, Feldman E, Goldman JM, Miller CB, Ottmann OG et al. Imatinib induces hematologic and cytogenetic responses in patients with chronic myelogenous leukemia in myeloid blast crisis: results of a phase II study. Blood 2002; 99: 3530–3539. | Article | PubMed | ISI | ChemPort |
  74. Ottmann OG, Druker BJ, Sawyers CL, Goldman JM, Reiffers J, Silver RT et al. A phase 2 study of imatinib in patients with relapsed or refractory Philadelphia chromosome-positive acute lymphoid leukemias. Blood 2002; 100: 1965–1971. | Article | PubMed | ISI | ChemPort |
  75. Melo JV, Barnes DJ. Chronic myeloid leukaemia as a model of disease evolution in human cancer. Nat Rev Cancer 2007; 7: 441–453. | Article | PubMed | ISI | ChemPort |
  76. Koptyra M, Falinski R, Nowicki MO, Stoklosa T, Majsterek I, Nieborowska-Skorska M et al. BCR/ABL kinase induces self-mutagenesis via reactive oxygen species to encode imatinib resistance. Blood 2006; 108: 319–327. | Article | PubMed | ISI | ChemPort |
  77. Nowicki MO, Falinski R, Koptyra M, Slupianek A, Stoklosa T, Gloc E et al. BCR/ABL oncogenic kinase promotes unfaithful repair of the reactive oxygen species-dependent DNA double-strand breaks. Blood 2004; 104: 3746–3753. | Article | PubMed | ISI | ChemPort |
  78. Sallmyr A, Tomkinson AE, Rassool FV. Up-regulation of WRN and DNA ligase IIIalpha in chronic myeloid leukemia: consequences for the repair of DNA double-strand breaks. Blood 2008; 112: 1413–1423. | Article | PubMed | ISI | ChemPort |
  79. Dierov J, Sanchez PV, Burke BA, Padilla-Nash H, Putt ME, Ried T et al. BCR/ABL induces chromosomal instability after genotoxic stress and alters the cell death threshold. Leukemia 2009; 23: 279–286. | Article | PubMed | ISI | ChemPort |
  80. Breedveld P, Beijnen JH, Schellens JH. Use of P-glycoprotein and BCRP inhibitors to improve oral bioavailability and CNS penetration of anticancer drugs. Trends Pharmacol Sci 2006; 27: 17–24. | Article | PubMed | ISI | ChemPort |
  81. Peng B, Lloyd P, Schran H. Clinical pharmacokinetics of imatinib. Clin Pharmacokinet 2005; 44: 879–894. | Article | PubMed | ISI | ChemPort |
  82. White DL, Saunders VA, Dang P, Engler J, Venables A, Zrim S et al. Most CML patients who have a suboptimal response to imatinib have low OCT-1 activity: higher doses of imatinib may overcome the negative impact of low OCT-1 activity. Blood 2007; 110: 4064–4072. | Article | PubMed | ISI | ChemPort |
  83. Burger H, van Tol H, Boersma AW, Brok M, Wiemer EA, Stoter G et al. Imatinib mesylate (STI571) is a substrate for the breast cancer resistance protein (BCRP)/ABCG2 drug pump. Blood 2004; 104: 2940–2942. | Article | PubMed | ISI | ChemPort |
  84. Thomas J, Wang L, Clark RE, Pirmohamed M. Active transport of imatinib into and out of cells: implications for drug resistance. Blood 2004; 104: 3739–3745. | Article | PubMed | ISI | ChemPort |
  85. Yong AS, Keyvanfar K, Hensel N, Eniafe R, Savani BN, Berg M et al. Primitive quiescent CD34+ cells in chronic myeloid leukemia are targeted by in vitro expanded natural killer cells, which are functionally enhanced by bortezomib. Blood 2009; 113: 875–882. | Article | PubMed | ISI
  86. Dillmann F, Veldwijk MR, Laufs S, Sperandio M, Calandra G, Wenz F et al. Plerixafor inhibits chemotaxis toward SDF-1 and CXCR4-mediated stroma contact in a dose-dependent manner resulting in increased susceptibility of BCR-ABL(+) cell to Imatinib and Nilotinib. Leuk Lymphoma 2009; 55: 1676–1686. | Article
  87. Jin L, Tabe Y, Konoplev S, Xu Y, Leysath CE, Lu H et al. CXCR4 up-regulation by imatinib induces chronic myelogenous leukemia (CML) cell migration to bone marrow stroma and promotes survival of quiescent CML cells. Mol Cancer Ther 2008; 7: 48–58. | Article | PubMed | ISI | ChemPort |
  88. Barnes DJ, Schultheis B, Adedeji S, Melo JV. Dose-dependent effects of Bcr-Abl in cell line models of different stages of chronic myeloid leukemia. Oncogene 2005; 24: 6432–6440. | Article | PubMed | ISI | ChemPort |